Immunoassay is an analytical technique widely used in medicine and in the biological sciences. The term "immunoassay" as used herein encompasses analytical methods for detecting, locating or quantifying biological substances by use of a label. Generally, a "label" is attached to a molecule of the substance of interest. The presence of the labeled molecule can then be detected by suitable means.
There are various types of immunoassay in common usage. In one type of immunoassay, a sample containing both an unknown and a labeled antigen of interest is incubated with an antibody specific for that antigen. If the unknown also contains the antigen, then both the labeled and unlabeled antigens compete for binding sites on the antibody. The antibody can be immobilized on a solid support, such as a test tube, glass beads, latex particles, or the like. Incubation is followed by a separation step in which the antigen bound to the antibody on the support is separated from unbound antigen. Through measurement of the amount of bound labeled antigen, the presence and/or quantity of similar, unlabeled antigen can be determined. Thus, the detected level of the labeled antigen (e.g. counts per minute of radioactivity) is an inverse function of the concentration of the unlabeled antigen.
A second type of immunoassay is known as sandwich immunoassay. In this method, an antibody rather than an antigen is labeled. A sample containing an unknown is incubated with an immobilized antibody. Antigens, if present in the sample, will bind to the antibody. After incubation, unbound material is removed by a separation step. In a second incubation with a solution of labeled antibody, the bound antigen is "sandwiched" between the immobilized antibody and the labeled antibody which adheres to the antigen. After a second separation, the amount of labeled antibody is determined. Detection of labeled antibody is indicative of the presence of antigen.
In general, a commonly used type of label is a radioactive substance, which can easily and accurately be detected. However materials labeled radioactively often have a short shelf life, both because of radioactive decay of the label and because radiation degrades the labeled molecule. Further, handling of radioactive substances entails risks to laboratory personnel.
In contrast to radioimmunoassay, luminometric immunoassay utilizes a chemiluminescent compound as the label. Such a compound is capable of undergoing a reaction (usually oxidative) in which light is a product. The light emission is measured by appropriate devices, and in certain cases, the light intensity is indicative of the quantity of labeled material. Known chemiluminescent substances suitable for use as immunoassays include luminol, isoluminol, and the various acridinium esters, for example, as noted in the prior art literature, and discussed in U.S. Pat. No. 5,395,938, to Ramakrishnan, which is commonly assigned with the present application.
Luminometric immunoassay procedures overcome many of the problems encountered with radioimmunoassay, namely risk to personnel and the short shelf life due to radioactive decay. Additionally, for example, luminometric immunoassay is easier to use, requires a shorter incubation time, solves problems related to safety, waste disposal, and regulatory compliance, has greater sensitivity, utilizes more stable reagents, and improves ease of manufacture and storage.
It is known in the prior art to employ an automated chemiluminescence immunoassay analyzer for assaying specimens. Such automated systems typically employ a set of two trigger reagents A and B which can trigger a chemiluminescent reaction, in labels such as either luminol or acridinium. The trigger reagents are sequentially injected into a receptacle, such as the well of a cuvette, when the cuvette is disposed in the measuring chamber of the analyzer. When the chemiluminescent reaction is initiated by the injection of the trigger reagents, the flash resulting from the oxidation of the label to its excited state, and its subsequent return to the ground state, which typically lasts about two seconds, is detected by the system integrated luminometer. This value is expressed in relative light units (RLU), and compared to a calibrated test standard in order to determine the amount of bound labeled antibody or antigen in the patient's blood.
Assays are available to test a variety of body functions, including, for example, the adrenal/pituitary system, anemia, bone and mineral metabolism, growth, thyroid, tumor markers, hypertension, neonatal conditions, and the reproductive system. It would be advantageous to be able to perform a plurality of such assays, of different types, on a single immunoassay instrument, in a single procedure, using a plurality of labels, and a plurality of triggering reagent sets to initiate a chemiluminescent reaction for each of the labels sequentially. It would further be advantageous to be able to inject the triggering reagents of each set in such a manner that the walls of the cuvette well are washed down and cross-contamination between trigger reagents exiting each of the exit ports of the trigger reagent injector is substantially eliminated.